Helper virus-free aav production

ABSTRACT

A method for the production of adeno-associated virus stocks and recombinant adeno-associated virus stocks that are substantially free of contaminating helper virus is described. The method utilizes transfection with helper virus vectors to replace the infection with helper virus used in the conventional method.

TECHNICAL FIELD

The present invention relates to methods, cells and vectors for theproduction of adeno-associated viral stocks that are substantially freeof helper virus.

BACKGROUND

Adeno-associated virus (AAV) is a defective member of the parvovirusfamily. The AAV genome is encapsidated as a single-stranded DNA moleculeof plus or minus polarity (Berns and Rose, 1970, J. Virol. 5:693-699;Blacklow et al., 1967, J. Exp. Med. 115:755-763). Strands of bothpolarities are packaged, but in separate virus particles (Berns andAdler, 1972, Virology 9:394-396) and both strands are infectious(Samulski et al., 1987, J. Virol. 61:3096-3101).

The single-stranded DNA genome of the human adeno-associated virus type2 (AAV2) is 4681 base pairs in length and is flanked by terminalrepeated sequences of 145 base pairs each (Lusby et al., 1982, J. Virol.41:518-526). The first 125 nucleotides form a palindromic sequence thatcan fold back on itself to form a “T”-shaped hairpin structure and canexist in either of two orientations (flip or flop), leading to thesuggestion (Berns and Hauswirth, 1979, Adv. Virus Res. 25:407-449) thatAAV may replicate according to a model first proposed by Cavalier-Smithfor linear-chromosomal DNA (1974, Nature 250:467-470) in which theterminal hairpin of AAV is used as a primer for the initiation of DNAreplication. The AAV sequences that are required in cis for packaging,integration/rescue, and replication of viral DNA appear to be locatedwithin a 284 base pair (bp) sequence that includes the terminal repeatedsequence (McLaughlin et al., 1988, J. Virol. 62:1963-1973).

At least three regions which, when mutated, give rise to phenotypicallydistinct viruses have been identified in the AAV genome (Hermonat etal., 1984, J. Virol. 51:329-339). The rep region codes for at least fourproteins (Mendelson et al., 1986, J. Virol 60:823-832) that are requiredfor DNA replication and for rescue from the recombinant plasmid. The capregion encodes AAV capsid proteins; mutants containing lesions withinthis region are capable of DNA replication (Hermonat et al., 1984, J.Virol. 51:329-339). AAV contains three transcriptional promoters (Carteret al., 1983, in “The Parvoviruses”, K. Berns ed., Plenum PublishingCorp., NY pp. 153-207; Green S and Roeder, 180, Cell 22:231-242,Laughlin et al., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5567-5571; Lusbyand Berns, 1982, J. Virol. 41:518-526; Marcus et al., 1981, Eur. J.Biochem. 121:147-154). The viral DNA sequence displays two major openreading frames, one in the left half and the other in the right half ofthe conventional AAV map (Srivastava et al., 1985, J. Virol.45:555-564).

AAV can be propagated as a lytic virus or maintained as a provirus,integrated into host cell DNA (Cukor et al., 1984, in “TheParvoviruses,” Berns, ed., Plenum Publishing Corp., NY pp. 33-66).Although under certain conditions AAV can replicate in the absence ofhelper virus (Yakobson et al., 1987, J. Virol. 61:972-981), efficientreplication requires coinfection with a helper virus, includingadenovirus (Atchinson et al., 1965, Science 194:754-756; Hoggan, 19865,Fed. Proc. Am. Soc. Exp. Biol. 24:248; Parks et al., 1967, J. Virol.1:171-180); herpes simplex virus (Buller et al., 1981, J. Virol.40:241-247) or cytomegalovirus, Epstein-Barr virus, or vaccinia virus.Hence the classification of AAV as a “defective” virus.

When no helper virus is available, AAV can persist in the host cellgenomic DNA as an integrated provirus (Berns et al., 1975, Virology68:556-560; Cheung et al., 1980, J. Virol. 33:739-748). Virusintegration appears to have no apparent effect on cell growth ormorphology (Handa et al., 1977, Virology 82:84-92; Hoggan et al., 1972,in “Proceedings of the Fourth Lapetit Colloquium, North HollandPublishing Co., Amsterdam pp. 243-249). Studies of the physicalstructure of integrated AAV genomes (Cheung et al., 1980, supra; Bernset al., 1982, in “Virus Persistence”, Mahy et al., eds., CambridgeUniversity Press, NY pp. 249-265) suggest that viral insertion occurs atrandom positions in the host chromosome but at a unique position withrespect to AAV DNA, occurring within the terminal repeated sequence.More recent work has revealed the AAV integration into the hostchromosome may not be random after all but is preferentially targeted toa site on chromosome 19 (Samulski 1993 Curr. Opinion in Genet. andDevel. 3:74-80). Integrated AAV genomes have been found to beessentially stable, persisting in tissue culture for greater than 100passages (Cheung et al., 1980 supra).

Although AAV is believed to be a human virus, its host range for lyticgrowth is unusually broad. Virtually every mammalian cell line(including a variety of human, simian, and rodent cell lines) evaluatedcould be productively infected with AAV, provided that an appropriatehelper virus was used (Cukor et al., 1984, in “The Parvoviruses”, Berns,ed. Plenum Publishing Corp., NY, pp. 33-66).

No disease has been associated with AAV in either human or animalpopulations (Ostrove et al., 1987, Virology 113:521-533) despitewidespread exposure and apparent infection. Anti-AAV antibodies havebeen frequently found in humans and monkeys. It is estimated that about70 to 80 percent of children acquire antibodies to AAV types 1, 2, and 3within the first decade; more than 50 percent of adults have been foundto maintain detectable anti-AAV antibodies. AAV has been isolated fromfecal, ocular, and respiratory specimens during acute adenovirusinfections, but not during other illnesses (Dulbecco and Ginsberg, 1980,in “Virology”, reprinted from Davis, Dulbecco, Eisen and Ginsberg's“Microbiology”, Third Edition, Harper and Row Publishers, Hagerstown, p.1059).

Recombinant Adeno-Associated Virus

Samulski et al., (1982, Proc. Natl. Acad. Sci. U.S.A. 79:2077-2081)cloned intact duplex AAV DNA into the bacterial plasmid pBR322 and foundthat the AAV genome could be rescued from the recombinant plasmid bytransfection of the plasmid DNA into human cells with adenovirus 5 ashelper. The efficiency of rescue from the plasmid was sufficiently highto produce yields of AAV DNA comparable to those observed aftertransfection with equal amounts of purified AAV virion DNA.

The AAV sequences in the recombinant plasmid could be modified, and then“shuttled” into eukaryotic cells by transfection. In the presence ofhelper adenovirus, the AAV genome was found to be rescued free of anyplasmid DNA sequences and replicated to produce infectious AAV particles(Samulski et al., 1982, Proc. Natl. Acad. Sci. 79:2077-2081; Laughlin etal., 1983, Gene 23:65-73; Samulski et al., 1983, Cell 33:134-143;Senaphthy et al., 1982, J. Mol. Biol. 179:1-20).

The AAV vector system has been used to express a variety of genes ineukaryotic cells. Hermonat and Muzyczka (1984, Proc. Natl. Acad. Sci.U.S.A. 81:6466-6470) produced a recombinant AAV (rAAV) viral stock inwhich the neomycin resistance gene (neo) was substituted for AAV capsidgene and observed rAAV transduction of neomycin resistance into murineand human cell lines. Tratschen et al. (1984, Mol. Cell. Biol.4:2072-2081) created a rAAV which was found to express thechloramphenicol acetyltransferase (CAT) gene in human cells. Lafare etal. (1988, Virology 162:483-486) observed gene transfer intohematopoietic progenitor cells using an AAV vector. Ohi et al. (1988, J.Cell. Biol. 107:304A) constructed a recombinant AAV genome containinghuman β-globin cDNA. Wondisford et al. (1988, Mol. Endocrinol. 2.32-39)cotransfected cells with two different recombinant AAV vectors, eachencoding a subunit of human thyrotropin, and observed expression ofbiologically active thyrotropin.

Several rAAV vector systems have been designed. Samulski et al. (1987,J. Virol. 61:3096-3101) constructed an infectious adeno-associated viralgenome that contains two XbaI cleavage sites flanking the viral codingdomain; these restriction enzyme cleavage sites were created to allownonviral sequences to be inserted between the cis-acting terminalrepeats of AAV. U.S. Pat. No. 4,797,368 relates to AAV vectors containedin a plasmid, capable of being packaged into AAV particles, andfunctioning as a vector for stable maintenance or expression of a geneor a DNA sequence in eukaryotic cells when under control of AAVtranscription promoter. Other AAV vectors and their uses are describedin U.S. Pat. No. 5,139,941 and WO 9413788.

Current methods for production of recombinant AAV (RAAV) viral stocksrequire infection of the host cell with a helper virus, like adenovirus,and transfection with the rAAV and with helper AAV DNA to supply intrans the essential AAV functions missing from the rAAV. Recent work hasaccomplished the production of rAAV stocks that are essentially free ofthe AAV helper (Samulski et al. 1989 J. Virol. 63:3822-3828). However,these rAAV stocks still contain virulent adenovirus or other helpervirus along with the rAAV virions. The method of the present inventionallows the production of recombinant AAV or wild type AAV in vivowithout a concomitant infection by adenovirus or other helper virus.Consequently the production of infectious helper virus contaminant isreduced or eliminated.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forproducing adeno-associated virus (AAV) stocks that are substantiallyfree of helper virus. The method can be used for production of eitherwild type AAV stocks or recombinant AAV (rAAV) stocks. The methodeliminates the conventional requirement for infection with helper virus.In particular, in the method of the present invention, the helper viralfunctions that are essential for productive infection by AAV areprovided by transfection of the host cell with helper virus vectors.Alternatively the helper virus vector may be provided from anextrachromosomal element in the host cell, or may be stably integratedinto the host cell chromosome, to provide a cell line which expressesthe helper viral functions essential for productive infection by AAV.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood by reference to thefollowing detailed description of the specific embodiments whenconsidered in combination with the drawings that form part of thepresent application, wherein:

FIG. 1. Schematic depiction of the conventional method for thepreparation of recombinant AAV stocks. pAAV/Ad and pAAV/β-gal plasmidsare transfected into 293 cells in the presence of Adenovirus infection.The infected/transfected cells yield two types of infectious viralparticles, Adenovirus virions and rAAV/β-gal virions.

FIG. 2. Schematic depiction of one embodiment of the method of thepresent invention. The Adenovirus genome is isolated and cleaved withrestriction enzyme XbaI. The large XbaI fragment of Adenovirus is thenused to transfect 293 cells together with pAAV/Ad and pAAV/β-galplasmids. The transfected cells yield only one type of infectious viralparticle, the rAAV/β-gal virion.

FIG. 3. Densitometer Scan of Dot Blot Hybridization of CsCl GradientFractions containing rAAV. The bar height indicates the intensity of thehybridization signal in genome equivalents (of rAAV DNA) per ml. Theopen bars indicate fractions from a rAAV preparation in which the celldisruption is accomplished by sonication. The filled-in bars representfractions from a rAAV preparation in which the cell disruption isaccomplished by freeze-thaw lysis followed by nuclease treatment.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Currently-used methods for production of AAV stocks and rAAV stocksrequire a concomitant infection with a helper virus, such as adenovirus,to achieve a productive infection by the AAV or rAAV (see FIG. 1). Thiscan result in a significant level of helper virus contamination of theAAV or rAAV stocks. The present invention solves this problem byeliminating the requirement for infection with helper virus. By themethod of the present invention, helper virus functions essential forproductive AAV infection are supplied in trans by transfection with ahelper virus vector that provides the viral functions necessary but thatcannot be packaged into a helper virus virion.

In general, the method of the present invention can be used to producerAAV stocks that are substantially free of helper virus by usingtransfection with a helper virus vector rather than infection withhelper virus as is used in the prior art methods. In another embodiment,the method of the present invention can be used to produce wild type AAVstocks that are substantially free of helper virus. The wt AAV stocksmay be made, for example, by infection with wt AAV virions ortransfection with infectious cloned AAV plasmids, in combination withtransfection with helper virus vector. In a further embodiment, thehelper virus vector may be stably incorporated into the host cell lineas an extrachromosomal element. In this embodiment, the essentialhelper-viral functions are provided from the extrachromosomal elementand additional transfection with helper virus vector is not required.

In particular, in one embodiment, the method of the present inventioncomprises:

(a) cotransfecting cells permissive for adenovirus-associated virusreplication with:

-   -   i) a recombinant adeno-associated virus vector which is capable        of being packaged into infectious AAV virions,    -   ii) a helper AAV vector which provides the AAV-viral functions        essential for the replication and packaging of said recombinant        adeno-associated virus vector into infectious AAV virions, and        -   iii) a helper virus vector which provides the helper-viral            functions essential for a productive adeno-associated virus            infection but which cannot itself be packaged into            infectious helper virus virions; and,

(b) collecting virions produced.

In another embodiment, the method of the present invention comprises:

(a) transfecting cells permissive for adenovirus-associated virusreplication with a helper virus vector which provides the helper-viralfunctions essential for a productive adeno-associated virus infectionbut which cannot itself be packaged into infectious helper virusvirions;

(b) infecting said cells with adeno-associated virus; and

(c) collecting virions produced.

In a third embodiment, the method of the present invention comprises:

(a) cotransfecting cells permissive for adenovirus-associated virusreplication, wherein said cells comprise an extrachromosomal elementcomprising a helper virus vector which provides the helper viralfunctions essential for a productive adeno-associated virus infection,with:

-   -   i) a recombinant adeno-associated virus vector which is capable        of being packaged into infectious AAV virions, and    -   ii) a helper AAV vector which provides the AAV-viral functions        essential for the replication and packaging of said recombinant        adeno-associated virus vector into infectious AAV virions; and

(b) collecting virions produced.

The present inventors have surprisingly found that productive infectionof AAV or rAAV can occur in host cells even in the absence of infectionby a helper virus if the helper viral functions essential for productiveAAV infection are supplied. By productive AAV infection is meant thatthe AAV or rAAV DNAs are replicated and packaged into infectious virionsin the host cell.

The helper virus vector of the present invention comprises DNA from anyof a number of helper viruses that are well known in the art (see, forexample, Berns and Labow, 1987, J. Gen. Virol. 68:601-614; Muzyczka,1992, Curr. Top. Microbiol. Immun. 158:97-129; Berns, 1990, Microbiol.Rev. 54:316-329). The helper viruses are those which support aproductive AAV infection. These viruses include, but are not limited to,adenovirus, herpes virus, cytomegalovirus, Epstein-Barr virus andvaccinia virus. The helper virus vectors of the present inventioncomprise DNA from a helper virus, which DNA provides for thehelper-viral functions essential for a productive AAV infection, but thevector itself cannot be packaged into infectious helper virus virions.Preferably, for the practice of the method of the present invention, ahelper virus vector may contain the entire genomic DNA of the helpervirus except for the cis-acting signals that function in the replicationand/or packaging of the helper virus. For many helper viruses thesecis-acting signals have been identified (see, for example, Sussenbach in“The Adenoviruses,” Ginsberg, H. ed. Plenum Press 1984, and thereferences therein; Fraenkel-Conrat, “Virology” 1982 Prentice-Hall, andthe references therein). The cis-acting signals can also be identifiedby the methods described herein; for example, by transfection of hostcells with helper virus genomic DNA containing various mutations orhelper virus DNA fragments and assaying for the production of helpervirus virions. More preferably for the practice of the presentinvention, the helper virus vectors comprise only those parts of thegenomic helper viral DNA that contain helper viral genes or othersequences that are essential for productive AAV infection. For example,the adenovirus E1, E2, E4, and VA gene products are known to beadenoviral functions essential for a productive adeno-associated virusinfection (Berns and Labow, 1987, J. Gen. Virol. 68:601-614).

Generally, the helper virus vector will contain helper viral DNAencoding all of the essential helper viral functions. However, when usedin combination with certain host cells which are able to express one ormore of the helper viral functions essential for productive AAVinfection, the helper virus vector may contain accordingly less of thehelper virus genomic sequence. For example, one preferable helper virusvector is the large XbaI fragment of adenovirus (Ad) dl309. Thisfragment is missing the left-most approximately 900 bases from theadenovirus genome. The left end of adenovirus dl309 contains thecis-acting signals necessary for replication and packaging of adenovirusbut also contains the promoter region for the Ad E1 gene. The Ad E1 geneproduct is essential for productive infection of AAV. However, the humancell line 293 expresses the Ad E1 gene so that use of 293 as a host fortransfection with the large XbaI fragment as a helper virus vector willprovide a full complement of essential helper viral functions. By usingthe methods described herein or other comparable methods well known inthe art, one of ordinary skill in the art can readily determine theparticular genomic sequences of any particular helper virus that areessential for productive AAV infection.

The particular helper virus DNA sequences to be included in the helpervirus vector may be determined by conventional mutation analysis of thehelper virus. For example, by using transfection with helper virusvectors containing various deletions or point mutations throughout thehelper viral genome, those regions of the helper viral genome whichprovide essential functions for productive infection of AAV can bedetermined. As an alternative to mutation analysis, various restrictionor other fragments of the helper virus genomic DNA can be assayed forthe presence of essential functions either by using the fragmentsdirectly for transfection or by cloning the fragments and using theclones for transfection. For any of these techniques, the helper viralDNA is transfected into host cells which are either transfected orinfected with AAV and the presence or absence of a productive AAVinfection is determined by conventional methods (for example, theinfectious center assay, McLaughlin et al. 1988). Alternatively, sinceproductive infection of AAV is dependent on the expression of AAV repand cap genes, the ability of the transfected helper viral DNA to inducethe expression of the AAV rep and cap genes (from a helper AAV vector)can be determined. As indicated above, many of the helper viral regionsthat encode functions essential for productive AAV infection are alreadyknown. Minimally, these known essential regions are included in thehelper virus vector.

The helper virus vector can be a DNA molecule in any convenient form,for example, an entire viral genome, restriction fragments of the viralgenome, plasmids or bacteriophages containing the helper viralsequences, or chemically or enzymatically synthesized DNA. The helpervirus vector DNA can be prepared by any appropriate method.

In particular, the large XbaI fragment from Ad dl309 (Jones and Shenk,Cell, 1979 17:683-689) may be used as a helper virus vector. Ad dl309contains a single XbaI site and cleavage with XbaI provides anapproximately 900 bp fragment from the left end and an approximately35,000 bp fragment from the right end. The larger fragment provides allof the helper viral functions essential for productive AAV infectionexcept for Ad E1. When the XbaI large fragment is used to transfecthuman 293 cells the Ad E1 is supplied by the 293 cells.

Helper AAV vectors and recombinant AAV vectors are well known in the art(see for example Muzyczka, 1992, supra; U.S. Pat. No. 5,139,941;WO9413788). Generally, recombinant AAV vectors contain the AAV invertedterminal repeats (or other sequences which enable the vector toreplicate and/or integrate and package, such as the double-D vectorsdescribed in WO9413788) ligated to a foreign (that is, non-AAV) gene orDNA sequence of interest. Recombinant AAV vectors that are suitable foruse with the present invention include, but are not limited to, psub201(Samulski 1987 J. Virol. 61:3096) and dl3-94 (McLaughlin et al. 1988 J.Virol. 62:1963). Generally, the helper AAV vectors express the AAVfunctions necessary to replicate and package the rAAV, including, forexample, the AAV rep and capsid genes. Helper AAV vectors suitable foruse with the present invention include but are not limited to pAAV/Ad(Samulski, 1989). The method of the present invention is not dependenton any particular rAAV vectors or helper AAV vectors and any suchsystems which are capable of producing infectious rAAV by conventionalmethods (that is, by coinfection with a helper virus) are suitable foruse in the method of the present invention.

Transfection may be performed by the DEAE-dextran method (McCutchen andPagano, 1968, J. Natl. Cancer Inst. 41:351-357), the calcium phosphateprocedure (Graham et al., 1973, J. Virol. 33:739-748) or by any othermethod known in the art, including but not limited to microinjection,lipofection, and electroporation. The amount of vector DNA used intransfection is approximately 0.2 to 10 μg of each DNA appropriate per10⁶ cells, but may vary among different DNA constructs and differentcell types. At the end of several hours to several days aftertransfection, the transfected cells are ruptured, for instance byfreeze-thaw techniques, sonication or dounce homogenization, and thevirions produced can be collected from the resulting lysate. The lysatecan be used directly for assay of the virion concentration or forinfection of recipient cells. Alternatively the virions in the lysatemay first be concentrated by centrifugation, for example, by densitygradient centrifugation in a CsCl gradient or by pelleting the virus athigh speeds.

Typically, for the method of the present invention, approximately 10host cells are transfected with between 0.2 ug and 10 ug each of ahelper virus vector, a helper AAV vector and a rAAV vector. The amountof DNA will depend on the particular vectors and cells used but ingeneral the molar ratios of the DNAs will be approximately 1:2:9 rAAVvector:helper virus vector:helper AAV vector, although variations inthis ratio for any particular combination of vectors can readily bedetermined by one of ordinary skill in the art by transfecting withvarying amounts of any particular vector and determining the optimumamount to obtain maximum virion yield. The amount of helper virus vectorwill generally be between 10³ and 10⁵ (in genome equivalents) times theamount of infectious virus (in plaque forming units) that would be usedfor an infection. For example, when the large XbaI fragment of Ad dl309is the helper virus vector, the amount of DNA used in the transfectionof 5×10⁶ cells is 2.6×10¹¹ genome equivalents. If the same amount ofcells are infected with adenovirus dl309, the optimum amount of virusfor infection (MOI=5) is 2×10⁷ plaque forming units. The cells areincubated for several hours to several days, preferably 24 to 72 hours,and then the cells are collected, lysed, and the virions are collectedand titered.

The host cells useful in the method of the present invention include anycells that are permissive for the replication of AAV, particularlymammalian cells, including, but not limited to, HeLa cells or human 293cells. It will be understood from the foregoing discussion that thechoice of host cell may depend in part on the particular helper virusvector employed. Cells having a latent AAV infection may also be used.

The titer of the AAV or rAAV virions obtained by the method of thepresent invention can be determined by methods identical to thosegenerally employed for determination of AAV viral titer for viral stocksprepared by conventional methods (see for example McLaughlin et al. 1988J. Virol. 62:1963; Dhawan et al, 1991). The particular method chosenwill depend on the particular genes or other DNA carried by the virion.For example, if the virion DNA carries the β-galactosidase gene (Lac Z),the titer may be estimated by transducing recipient cells and measuringthe frequency of expression of the β-galactosidase gene in thetransductants (see for example Dhawan et al. Science 254:1509 1991).Typically, the method of the present invention provides viral titers forrAAVs that are comparable to or higher than titers provided byconventional methods (that is, using helper virus infection).Surprisingly, the use of transfection with helper virus DNA rather thaninfection with helper virus does not result in a significant reductionin virus yield, presumably because both systems are limited by theefficiency of transfection of the rAAV plasmid and the helper AAVplasmid. Typically, the yields for wt AAV produced by the present methodis lower than that generally obtained by more conventional methods butthe wt AAV produced is substantially free of helper virus.

The method of the present invention provides rAAV or AAV stocks that aresubstantially free of helper virus. By substantially free of helpervirus is meant that the viral stocks produced by the method of thepresent invention produce no detectable cytopathic effect (CPE) on cellsthat are susceptible to helper virus infection. The cytopathic effectmay be conveniently determined by infecting 4×10⁵ appropriate host cellswith 10 ul of a 1 to 1000 dilution of a AAV viral stock produced by themethod of the present invention containing from 106 to 108 AAV per ml.Any cells that are susceptible to infection by the helper virus fromwhich the particular helper virus vector is derived are suitable fordetermining the CPE, although, conveniently, the same kind of cells willbe used for determination of CPE as for the production of the AAV orrAAV stocks. The infection is allowed to proceed for 1 hr, the medium isreplaced as appropriate and the cells are observed for any clearance. Ifno CPE is observed after 48 hrs, the AAV viral stocks are substantiallyfree of helper virus. Other methods for determination of CPE are usefulin the method of the present invention and are well known in the art(see, for example, Agha et al. 1988 J. Med. Virol. 26:85-92)

In another embodiment of the method of the present invention, cells aretransfected with a helper virus vector and infected with wild type (wt)AAV to produce wt AAV virions free of contaminating helper virus. Thetransfection and infection are carried out by procedures that are wellknown in the art and are described above. Alternatively, cells arecotransfected with the helper virus vector and an infectious AAVplasmid, for example, pSM620 (Samulski, 1982). Either of these methodswill provide wt AAV stocks that are substantially free of helper virusas described above for rAAV stocks.

In a further embodiment of the present invention, the helper virusvector may be present on an extrachromosomal element (such as amini-chromosome or episome) in the host cell. Such extrachromosomalelement is stably maintained in the host cell and provides the helperviral functions essential for a productive AAV infection without theneed for transfection with the helper virus vector each time. Mammalianextrachromosomal elements are known, for example the Epstein Barr virus(EBV) based nuclear episome (Margolski, 1992, Curr. Top. Microbiol.Immun. 158:67-95) and can be readily prepared from the helper virusvector by well known methods, for example, Giraud et al. (Proc. Natl.Acad. Sci., 1994, 91:10039-10043). Preferably, the extrachromosomalelement containing the helper virus vector will be present in from oneto 100 copies per cell. Alternatively, the helper virus vector can bestably integrated into the chromosome of a host cell to produce a cellline which expresses the helper viral functions essential for productiveAAV infection. Cell lines in which the helper virus vector is present onan extrachromosomal element or stably integrated into the chromosomalDNA can be identified and isolated by transfecting an appropriate hostcell with the helper virus vector and selecting cloned cell lines whichcan support the productive infection of AAV. Such cell lines can bedetermined by techniques that are well known in the art including, amongothers, transfecting with a helper AAV vector and assaying for theinduction of the AAV rep and cap genes, transfecting with a rAAV andassaying for the replication of the rAAV genome or transfecting withinfectious AAV plasmids and assaying for the production of infectiousAAV virions, and any combinations of these techniques. The use ofinducible promoters in the extrachromosomal element or the integratedhelper virus vector to regulate the expression of the essential helperviral functions is desirable to prevent the premature expression of thehelper viral genes, which may have a deleterious effect on the hostcell. Appropriate inducible promoters include, but are not limited to,the mouse metallothionein promoter, heat shock promoters (Wurm et al.PNAS 1986 83:5414-5418) glucocorticoid inducible promoters (Heynes etal. PNAS 1981 78:2038; Lee et al. Nature 1991 294:228) and well as thetranscriptional activator domains described in Deuschle et al. (Mol.Cell. Biol. 1995 15:1907-1914).

Specific examples of the steps described above are set forth in thefollowing examples. However, it will be apparent to one of ordinaryskill in the art that many modifications are possible and that theexamples are provided for purposes of illustration only and are notlimiting of the invention unless so specified.

EXAMPLES Example 1 Preparation of Adenovirus Genomic DNA

Host cells are infected with adenovirus at a multiplicity of infection(MOI) of 5 and harvested at clear CPE. Cells are pelleted bycentrifugation at 1000 rpm for 5 min. For every five 10 cm plates ofcells used, the cell pellet is resuspended in 5 ml of Tris-saline(0.025M Tris pH 7.4, 0.14 M NaCl, 30 mM KCl, 7 mM Na₂HPO₄, 6 mMdextrose). The resuspended pellet is subjected to freeze-thaw threetimes. The cell debris is removed by centrifugation and the supernatantis layered onto a CsCl step gradient (1.4 g/ml lower, 1.2 g/ml upper).The gradients are run in an SW41 rotor for 1 hr at 30,000 rpm. The lowervirus band is removed and adjusted to 1.34 g/ml and centrifuged at40,000 rpm for 24 hr in an SW41 rotor. The lower virus band is removedand dialyzed against 10 mM Tris (pH 7.5), 1 mM EDTA, 200 mM NaClfollowed by treatment with 200 ug/ml proteinase K, 0.5% SDS for 1 hr at37° C. The dialysate is extracted twice with two volumes of phenol andonce with two volumes of chloroform:isoamyl alcohol (24:1). The DNA isprecipitated with 1/10 volume 3 M sodium acetate and 2.5 volumes ofethanol.

Example 2 Preparation of Large XbaI Fragment of Adenovirus dl309 DNA

Adenovirus dl309 genomic DNA is isolated as described in Example 1. Thepurified Ad DNA (50 ug) is then incubated with 50 units of restrictionenzyme XbaI as suggested by the manufacturer until the digestion iscomplete. The XbaI digested DNA is separated by electrophoresis on a0.8% agarose gel (Maniatis et al. 1982, Molecular Cloning: a laboratorymanual. Cold Spring Harbor Laboratories) and the larger fragment (35,000bp) is excised from the gel. The agarose is solubilized using gelase (5Prime-3 Prime) and the DNA is precipitated with ethanol and resuspendedin water.

Example 3 Preparation of Infectious rAAV/β-Gal by Transfection withAdenovirus Dl309 Large XbaI Fragment

293 cells (Graham et al. 1977 J. Gen. Virol. 36:59-72) were passed into10 cm tissue culture plates at a density of 9.09×10⁴ cells/cm². Thefollowing day the cells were transfected using the CaPO₄ precipitatemethod (Gibco-BRL) for 12 hours. For each 10 cm dish the followingamount of DNAs were used: pAAV/β-gal (1 μg) (pAAV/β-gal is the same aspAB11 described in Goodman et al. 1994 Blood 84:1492-1500), pAAV/Ad (9μg), large XbaI fragment of adenovirus dl309 DNA (10 μg). At the end ofthe 12 hours the media was replaced with DMM+10% FCS, and the cells wereallowed to incubate for an additional 48 hours. At the end of theincubation the cells were collected, lysed by sonication, and theresulting lysate, containing the infectious virions was assayed forβ-galactosidase activity upon subsequent infection of either 293 or HeLacells (blue staining nuclei are indicative of packaged, infectiousrAAV). The cells were infected with various amounts of the lysate andfixed and stained 24 hours later. The presence of successfully packagedrAAV was evident by the number of blue staining nuclei. In additionthere was no evidence of cytopathic effect (CPE) which is indicative ofan absence of any contaminating Ad.

Example 4 Dot Blot Assay for Determination of Viral Titer

Ten 10 cm dishes of 293 cells are transfected as in Example 3. 48 hrafter transfection, the cells are collected, lysed either by sonicationor by freeze-thaw followed by treatment with RNaseA and DNaseI, and celldebris is removed by centrifugation. The virus is precipitated byaddition of an equal volume of saturated ammonium sulfate. Theprecipitate is resuspended in a CsCl solution (density=1.4 g/ml) andcentrifuged for 48 hr in a SW41 rotor at 40,000 rpm. The gradient isdripped and 0.4 ml fractions are collected. The fractions are assayedfor DNA according to the following procedure.

5 ul of CsCl gradient fraction is mixed with 2 ul RNase (1 mg/ml), 2 ulDNase (1 mg/ml), 1 ul 1 M CaCl₂, 1 ul 1 M MgCl₂ and 189 ul 50 mM Tris(pH 8). The mixture is incubated at 37° C. for 30 min and then 2 ul 0.5M EDTA (pH8), 4 ul 0.25 M EGTA (pH 8) and 10 ul 10% sarcosine are added.The mixture is heated to 70° C. for 10 min and then cooled to 37° C. 20ul of proteinase K (10 mg/ml) is added and incubated for 2 hrs at 37° C.40 ul 5 M NaOH, 20 ul 0.5 M EDTA (pH8) and 224 ul of water are added andthe samples are applied to separate wells of a dot blot device. The dotblot is hybridized by conventional procedures to the appropriate AAV (orhelper virus) probe and the amount of DNA is determined by comparison tostandards. FIG. 3 shows a densitometer scan of the dot blot of CsClgradient fractions from two different preparations of rAAV. The openbars indicate the amount of rAAV DNA (in genome equivalents/ml) ingradient fractions when the cells are disrupted by sonication; thefilled-in bars indicate the amount of rAAV DNA in gradient fractionswhen the cells are disrupted by freeze-thaw followed by treatment with acombination of RNaseA and DNaseI.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. A method for producing substantially helper-virus free stocks ofrecombinant adeno-associated virus comprising: (a) cotransfecting cellspermissive for adenovirus-associated virus replication with: i) arecombinant adeno-associated virus vector which is capable of beingpackaged into infectious AAV virions, ii) a helper AAV vector whichprovides the AAV-viral functions essential for the replication andpackaging of said recombinant adeno-associated virus vector intoinfectious AAV virions, and iii) a helper virus vector which providesthe helper-viral functions essential for a productive adeno-associatedvirus infection but which cannot itself be packaged into infectioushelper virus virions; and, (b) collecting virions produced.
 2. Themethod of claim 1 wherein said helper virus vector is an adenovirusvector.
 3. The method of claim 1 wherein said helper virus vectorcomprises the large XbaI fragment of adenovirus.
 4. The method of claim3 wherein said cells are human 293 cells.
 5. The method of claim 1wherein said helper AAV vector is incapable of being packaged intoinfectious AAV virions.
 6. A method for producing substantiallyhelper-virus free stocks of adeno-associated virus comprising: (a)transfecting cells permissive for adenovirus-associated virusreplication with a helper virus vector which provides the helper-viralfunctions essential for a productive adeno-associated virus infectionbut which cannot itself be packaged into infectious helper virusvirions; (b) infecting said cells with adeno-associated virus; and (c)collecting virions produced.
 7. A method for producing substantiallyhelper-virus free stocks of adeno-associated virus comprising: (a)transfecting cells permissive for adenovirus-associated virusreplication with a helper virus vector which provides the helper-viralfunctions essential for a productive adeno-associated virus infectionbut which cannot itself be packaged into infectious helper virusvirions; (b) transfecting said cells with infectious adeno-associatedvirus DNA; and (c) collecting virions produced.
 8. Substantiallyhelper-virus free stocks produced by the method of claim
 1. 9.Substantially helper-virus free stocks produced by the method of claim6.
 10. Substantially helper-virus free stocks produced by the method ofclaim
 7. 11. A method for producing substantially helper-virus freestocks of recombinant adeno-associated virus comprising: (a)cotransfecting cells permissive for adenovirus-associated virusreplication, wherein said cells comprise an extrachromosomal elementcomprising a helper virus vector which provides the helper viralfunctions essential for a productive adeno-associated virus infection,with: i) a recombinant adeno-associated virus vector which is capable ofbeing packaged into infectious AAV virions, and ii) a helper AAV vectorwhich provides the AAV-viral functions essential for the replication andpackaging of said recombinant adeno-associated virus vector intoinfectious AAV virions; and (b) collecting virions produced. 12.Substantially helper-virus free stocks produced by the method of claim11.
 13. A cell line produced by the method comprising the steps of (a)transfecting cells permissive for adenovirus-associated virusreplication with: a helper virus vector which provides the helper-viralfunctions essential for a productive adeno-associated virus infectionbut which cannot itself be packaged into infectious helper virusvirions; and, (b) selecting a cell line wherein said helper virus vectoris present on an extrachromosomal element.
 14. A cell line produced bythe method comprising the steps of (a) transfecting cells permissive foradenovirus-associated virus replication with: a helper virus vectorwhich provides the helper-viral functions essential for a productiveadeno-associated virus infection but which cannot itself be packagedinto infectious helper virus virions; and, (b) selecting a cell linewherein said helper virus vector is stably integrated into thechromosomal DNA.